HETEROCYCLES

Paper | Regular issue | Vol. 87, No. 5, 2013, pp. 1029-1037
Received, 26th February, 2013, Accepted, 27th March, 2013, Published online, 2nd April, 2013.
DOI: 10.3987/COM-13-12691

■ New Diketopiperazine Derivatives from Culture Broth of Staphylococcus sp. Isolated from Corallina officinalis Lineaus

Amgad I. M. Khedr, Isao Kouno, Takashi Tanaka, and Koji Yamada*

Graduate school of Biomedical Sciences, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852-8521, Japan

Abstract

Two new diketopiperazine derivatives, staphyloamides A (1), and B (2), have been isolated from the culture broth of Staphylococcus sp. (No. P-100826-4-6) derived from Corallina officinalis Lineaus, along with the known cyclo (L-pro-D-phe) (3), cyclo (D-6-Hypro-L-phe) (4), cyclo (L-pro-L-val) (5), cyclo (L-pro-L-phe) (6), cyclo (L-pro-L-tyr) (7), cyclo (L-pro-L-leu) (8), cyclo (L-pro-L-ala) (9), and bacillusamide B (10). These structures were elucidated by extensive spectroscopic methods. Antimicrobial activities of compounds 3–10 were done.

INTRODUCTION
Natural products are a major resource for drug development. A large number of plants, microbes, and marine animals have been examined for bioactive secondary metabolites.1 Marine microorganisms are of considerable current interest as a new and promising source of biologically active compound, many of which can be used for drug development.2,3 During the past two decades, research on marine bacteria has highlighted the tremendous potential of this microorganism as a source of new bioactive secondary metabolites.4,5 Recently, special interest has pointed to diketopiperazines which are an important class of compounds displaying a variety of biological effects, such as antimicrobial, herbicidal, antiviral, immunosuppressive and cytotoxic activities.6-8 As a part of ongoing effort to discover biologically active metabolites from marine bacteria, Staphylococcus sp. (No. P-100826-4-6) was selected from our screening program for further studies. It has been observed that culture broth of Staphylococcus sp. (No. P-100826-4-6) derived from Corallina officinalis Lineaus family Corallinaceae, showed antifungal activity against Aspergillus niger, Penicillium crustosum and Schizophyllum commune. Therefore, we have researched the active constituents of this bacterium. The marine bacterium Staphylococcus sp. was obtained from Corallina officinalis Lineaus family Corallinaceae, collected in Nagasaki Shitsu coast of Japan in 2010. The strain was cultured at 25 ºC on rotary shakers using a seawater-based medium. The fermentation broth (32L) was successively partitioned with EtOAc to give an EtOAc extract (5.2 g) and the aqueous layer was subjected to Diaion HP-20 using H2O, 60% MeOH, 100% MeOH and acetone to give 60% MeOH (28.3 g), 100% MeOH (8 g) and acetone (3.9 g) elution fractions respectively. Of them, the EtOAc extract and 100% MeOH elution fraction showed antifungal activity against the fungi stated above. The EtOAc extract and 100% MeOH elution fraction were subjected to Sephadex LH-20, silica gel column chromatography and octa decyl silyl (ODS) column chromatography followed by reversed phase HPLC. As a result, two new diketopiperazines, staphyloamides A (1) and B (2), have been isolated along with eight known compounds: 3,9 4,10 5,11 6,12 7,13 8,14 9,15 and 1016 (Figure 1). The structures of these compounds were elucidated by using extensive spectroscopic methods.

RESULTS AND DISCUSSION
Staphyloamides A (1) was obtained as a white amorphous powder. The molecular formula was assigned as C11H19N2O3 by HR-FAB-MS (m/z 227.1396 [M+H] +, calcd 227.1391, Δ + 0.5 mmu), indicating 4 degrees of unsaturation. The IR spectrum of 1 suggested the presence of a hydroxy group (3229 cm-1) and amide carbonyl groups (1699 cm-1 and 1652 cm-1). The 1H-NMR spectrum showed signals for an isobutyl group [δH 0.79 (3H, t, J= 7.5 Hz, H-12), 1.20 (3H, d, J= 6.8 Hz, H-13), 1.37, 1.93 (1H, each m, H-11) and 2.51 (1H, m, H-10)] in addition to an amide proton [δH 9.54 (1H, brs, H-8)] (Table 1). The 13C-NMR spectrum displayed 11 carbon signals, including two methyl carbons [δC 11.0 (C-12), 16.0 (C-13)], three methylene carbons [δC 19.9 (C-4), 25.8 (C-11), 38.1(C-5)], one methylene carbon bearing nitrogen [δC 45.9 (C-3)], one methine carbon [δC 40.1 (C-10)], one methine carbon bearing nitrogen [δC 63.0 (C-9)], one quaternary carbon bearing oxygen [δC 87.4 (C-6)] and two carbonyl carbons [δC 167.4 (C-1), 169.5 (C-7)] (Table 1). These data together with degree of unsaturation revealed that 1 contained two rings in the molecule, thus 1 was found to have a bicyclic skeleton. Analysis of 1H-1H COSY spectrum of 1 showed extended 1H-1H spin system from H-9 to H3-12 via H-10 and H-11, as well as a vicinal correlation from H-10 to H3-13, in addition to 1H-1H COSY correlations of H-3 – H-5. The hetero nuclear multiple bond correlation (HMBC) of NH-8 to C-1 and C-10 were observed, as well as H3-13 to C-9, C-10, C-11, and H-9 to C-10, in addition to the correlation of H-12 to C-10 and C-11and H-9 and NH-8 to C-1 carbonyl, giving rise to an isoleucine moiety (b) (Figure 2), Moreover HMBC correlations of H-5 to C-3, C-6, and C-4 indicated the presence of proline moiety (a) (Figure 2). The connectivity of these two rings was revealed by HMBC correlation of NH-8 to C-6, C-1 and C-10 (Figure 2). The relative stereochemistry of C-6 and C-9 of 1 was confirmed on the basis of comparing the 13C- NMR data of 1 with those of known compound, notoamide M and other known compounds containing hydroxyproline moiety.10,16-19 Because the chemical chifts of C-3, C-4, C-5, C-6 and C-7 showed the same data with those of notoamide M, the relative stereochemistry of C-6 and C-9 as shown in Figure 1. The absolute configuration of the amino acid of 1 was determined by acid hydrolysis and Marfay’s method,20 using standard amino acids. The absolute configuration of the isoleucine was determined as D-form (Table 3). Thus, the absolute configuration was defined as 6 R, 9 R.
Staphyloamide B (2) was obtained as white amorphous powder. The molecular formula was assigned as C10H17N2O3 by HR-FAB-MS (m/z 213.1266 [M+H]+, calcd 213.1240, Δ+2.6 mmu), indicating 4 degrees of unsaturation. The IR spectrum of 2 suggested the presence of a hydroxy group (3244 cm-1) and amide carbonyl groups (1700 cm-1 and 1652 cm-1). The 1H-NMR spectrum showed signals for an isopropyl group [δH 1.12 (3H, d, J= 7.1 Hz, H-12), 1.16 (3H, d, J= 7.1 Hz, H-11), 2.83 (1H, dsept, J= 7.1, 4.6 Hz, H-10)] and amide proton [δH 8.73 (1H, brs, H-8)] (Table 2). The 13C-NMR spectrum displayed 10 carbon signals, including two methyl carbons [δC 16.8 (C-12), 18.8 (C-11)], two methylene carbons [δC 20.5 (C-4), 37.1 (C-5)], one methylene carbon bearing nitrogen [δC 45.3 (C-3)], one methine carbon [δC 29.2 (C-10)], one methine carbon bearing nitrogen [δC 60.7 (C-9)], one quaternary carbon bearing oxygen [δC 88.0 (C-6)] and two carbonyl carbons [δC 167.5 (C-1), 170.4 (C-7)] (Table 2). These data showed the presence of two amide groups and thus 2 was found to have a bicyclic skeleton in the same manner as 1. Detailed analysis of 2D NMR spectral data such as COSY and HMBC spectrum showed the presence of two partial structures including 6-hydroxyproline moiety (a) and valine moiety (b) (Figure 2). Furthermore, the connectivity of these partial structures was revealed by HMBC correlation of NH-8 to C-6, C-1 and H-5 to C-7. The 1H- and 13C-NMR signals of compound 2 and previously isolated compound bacillusamide B (10)16 showed close correspondence, except the chemical shift of H-9 [2: δH 4.50 (1H, d, J= 2.5 Hz), 10: 3.87 (1H, dd, J= 6.8, 4.0 Hz)]. This fact described that 2 was diastereoisomer to bacillusamide B (10) at position C-9 as shown in Figure 1.

The absolute configuration of the valine resdue was determined as L-form (Table 3). Thus, the absolute configuration was defined as 6 R, 9 S.

The antimicrobial activities of compounds 3–10 were tested for the growth inhibition of 11 microbes including gram positive, negative bacteria and fungi using a paper disk method.21 The growth inhibition was studied in a concentration of 100 and 50 μg /disk. As result, compound 4 and 8 slightly showed an inhibition circle against Schizophyllum commune, Staphylococcus aureus subsp. aureus, and Escherichia coli, while other compounds did not show antimicrobial activity at this concentration. The bioactivity of compounds 1 and 2 couldn’t be done due to small amounts.

EXPERIMENTAL
General
IR spectra were obtained with JASCO FT/IR-410 spectrophotometers. Optical rotations were measured with a JASCO DIP-370 digital polarimeter. 1H- and 13C-NMR, 1H-1H COSY, NOESY, HSQC and HMBC spectra were recorded with a Unity plus 500 spectrometer (Varian Inc., U.S.A.) operating at 500 MHz for 1H, and 125 MHz for 13C, respectively.1H-NMR chemicals hifts are expressed in δ values referring to the solvent peak δH 7.19 for pyridine-d5, and coupling constants are expressed in Hz. 13C-NMR chemical shifts are expressed in δ values referring to the solvent peak δC 123.5 for pyridine-d5. Sephadex LH-20 (Pharmacia Fine Chemical Co.Ltd),Wakogel C-300 (Wako Pure Chemical Industries, Ltd, Japan, 45~75 μm) and Cosmosil 5C18-140 PREP (Nakarai tesque, No.379-34) were used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and precoated RP-18 F254s plates (Merck) were used for thin-layer chromatography (TLC) analysis. FAB-MS were recorded on JMS DX-303 spectrometer (JEOL Ltd., Japan), and m-nitrobenzyl alcohol or Magic bullet as a matrix. Preparative HPLC was peroformed on a Develosil C-30-UG-5 (250 x 4.6 mm i.d Nomura Chemical Co., Aichi, Japan) and a Wakosil-II 5sil-100 (150 x 4.6 mm “B” Wako pure chemical industries, Ltd, Japan) at a flow rate of 1.5 mL/min, equipped with a TOSOH RI-8020 detector and a JASCO BIP-I HPLC pump.

Bacterial Material and Fermentation
The marine Staphylococcus sp. (strain number P-100826-4-6) was isolated from Corallina officinalis Linnaeus family Corallinaceae, collected in Nagasaki Shitsu coast of Japan in 2010. The subcultures of the bacterium are deposited at the Garden for Medicinal Plants, Graduated School of Biomedical Sciences, Nagasaki University. The bacterium was grown in a seawater-based medium (D-glucose 1%; polypeptone 0.5%; yeast extract 0.3%; KH2PO4 0.3%; MgSO4 0.1%; pH 7.5), using rotary-shaking at 120 rpm for 28 days at 25 °C. The culture broth (32L) was sonicated then filtrated.
Extraction and Isolation
The filtered broth was extracted by EtOAc (10 L x3). The EtOAc extract was concentrated under a reduced pressure to dryness to produce EtOAc extract (5.2 g). The aqueous layer were chromatographed on Diaion HP-20 column eluting with H2O, 60% MeOH, 100% MeOH and acetone respectively to give elution fractions of 60% MeOH (28.3 g), 100% MeOH (8.0 g) and acetone (3.9 g). The dried residue of EtOAc extract (5.2 g) was subjected to silica gel column chromatography (CHCl3 - MeOH = 10:0 ~ 0:10) to yield 25 fractions. The 13th fraction (1.1 g) was chromatographed on Sephadex LH-20 (CHCl3 - MeOH = 1:1) to yield four fractions (fractions A – D). Fraction B (143 mg) was subjected to silica gel column chromatography (n-hexane- acetone = 10:0 ~ 0:10) to yield six fractions. Fraction B-2 was subjected to reversed phase HPLC Develosil C-30 (30% MeOH - H2O) to give compounds 5 (12 mg) and 9 (20 mg). Fraction D (380 mg) was subjected to silica gel column chromatography (n-hexane - acetone = 10:0 ~ 0:10 followed by (acetone - MeOH = 5:5 ~ 0:10) to yield four fractions (fractions D-1 – D-4). Fraction D-2 was purified on Sephadex LH-20 (CHCl3 - MeOH = 1:1) to give compound 10 (10 mg). Fraction D-1 (30 mg) was subjected to reversed phase HPLC Wakosil 5C-18 (40% MeOH - H2O) to give compounds 2 (2.3 mg) and 1 (1.7 mg). The 100% MeOH elution fraction (8.0 g) was dissolved in a mixture of CHCl3 - MeOH - H2O = 5:5:1 to give soluble part (4.7 g) and an insoluble part (2.3 g). The soluble part was subjected to Sephadex LH-20 (CHCl3 - MeOH = 1:1) to yield seven fractions (fractions from A – G). Fraction D (587 mg) was chromatographed on ODS column chromatography (MeOH - H2O = 20:80 ~100:0) to give seven fractions. Fraction D-3 (105 mg) was chromatogaphed on silica gel column chromatography (CHCl3 - MeOH = 10:0 ~ 0:10) to give four fractions (fractions D-3a – D-3d). Fraction D-3a (73 mg) was subjected to Sephadex LH-20 (100% MeOH) followed by silica gel column chromatography (CHCl3 - MeOH = 99:1) to give compounds 8 (4 mg) and 6 (37 mg). Fraction D-3c (13 mg) was subjected to reversed phase HPLC Wakosil 5C-18 (40% MeOH - H2O) to give compounds 3 (4 mg) and 4 (1.7 mg). Fraction E (465 mg) was chromatographed on silica gel column chromatography (CHCl3 - MeOH = 10: 0 ~ 0:10) to give eight fractions. Fraction E-5 (32 mg) was subjected to Sephadex LH-20 (CHCl3 -MeOH = 1:1) followed by ODS column chromatography (20% MeOH - H2O) to yield compound 7 (13 mg).
Staphyloamide A (1): White amorphous powder [α]27D­ - 21.5 (c 0.085, MeOH); IR νmax (dry film) 3229, 2974, 1699, 1652, 1429 cm-1, 1H- and 13C-NMR data (see Table 1); (+) FAB-MS m/z: 227 [M+H]+, (+) HR- FAB-MS m/z: 227.1396 [M+H]+ (calcd for C11H19N2O3, 227.1391, Δ + 0.5 mmu).
Staphyloamide B (2): White amorphous powder [α]27D­ -146.5 (c 0.1, MeOH); IR νmax (dry film) 3244, 2924, 1700, 1652, 1436 cm-1, 1H- and 13C-NMR data (see Table 2); (+) FAB-MS m/z: 213 [M+H]+, (+) HR- FAB-MS m/z: 213.1266 [M+H]+ (Calcd for C10H17N2O3, 213.1240, Δ + 2.6 mmu).
Determination of Amino Acids Configuration
A sample of 1 or 2 (0.5 mg) was hydrolyzed in 6 N HCl (1 mL) at 110 °C for 16 h. After concentration to dryness, the residue was dissolved in 50 μL of pure H2O and 40 μL of 1M NaHCO3 aq. and 100 μL of 1% of Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide (FDLA) in acetone were added. The mixture was heated at 37 °C for 1 h and 20 μL of 1N HCl was added. The solution was evaporated to dryness to furnish a yellow solid. The residue was dissolved in H2O/MeCN (500 μL) and analyzed by reversed-phase HPLC (Mightysil RP-18 (250 x 4.6 mm i.d., Kanto Chemical Co., Inc.), mobile phase; 40% MeCN–H2O, flow rate; 1 mL/min, detection; UV 340 nm). and comparing with standard L- and D- amino acid in Table 3.
Antimicrobial activity assay
Activities of compounds were tested by paper disk methods against Bacillus subtilis subsp. subtilis, Staphylococcus aureus subsp. aureus, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger, Penicillium crustosum, Schizophyllum commune，Trichophyton concentrieum，Saccharomyces cerevisae, Serratia marcescens subsp. marcescens and Candida albicans with 100 and 50 μg/ disk as result, compound 4 and 8 slightly showed an inhibition circle against Schizophyllum commune, Staphylococcus aureus subsp. aureus, and Escherichia coli, while other compounds showed no inhibition circle.

ACKNOWLEDGEMENTS
We are grateful to Mr. M. Inada and Mr. N. Yamaguchi of the Scientific Support Section of Joint Research Center, Nagasaki University, for 1H-NMR, 13C-NMR and MS measurements. This work was supported in part by a Grant-in-Aid for Scientific Research No. 23590008 from the Japan Society for the Promotion of Science, which is gratefully acknowledged.

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